Inhibition of human telomerase by a G-quadruplex-interaction compound

ABSTRACT

Certain non-nucleoside compounds that will selectively inhibit telomerase by targeting the nucleic add structures, such as G-quadruplexes, that may be associated with human telomeres or telomerase have been identified. Inhibition of human telomerase by two perylenetetracarboxylic acid diimides and a carbocyanine has been demonstrated.  1 H-NMR studies have evidenced the stabilization of a G-quadruplex by the perylenetetracarboxylic acid diimide compounds and provided evidence that these and structurally related compounds inhibit the telomerase enzyme by a mechanism consistent with interaction with G-quadruplex structures.

The present application is a division of copending U.S. application Ser.No. 09/730,893, filed Dec. 5, 2000, which is a division of U.S.application Ser. No. 09/244,675, filed Feb. 4, 1999, and now issued asU.S. Pat. No. 6,156,763, which claims the priority of U.S. ProvisionalPatent Application Serial No. 60/073,629, filed Feb. 4, 1998. The entiredisclosure of each of these applications is incorporated herein byreference without disclaimer.

The government may own rights in the present invention pursuant tocontract number U19CA-67760-02, and contract number NCDDG, CA67760 fromthe National Cancer Institute, and contract number CA49751 and contractnumber CA77000 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

I. Field of the Invention

This invention relates to the field of cancer therapy. The inventionalso relates to screening methods for identifying pharmacologicallyactive compounds that may be useful for treating proliferative diseases.More particularly, the inventors have identified non-nucleoside moleculecompounds that interact with specific DNA structures and which inhibithuman telomerase.

II. Description of Related Art

Cancer, which is a cell proliferative disorder, is one of the leadingcauses of disease, being responsible for 526,000 deaths in the UnitedStates each year (Boring et al., 1993). For example, breast cancer isthe most common form of malignant disease among women in Westerncountries and, in the United States, is the most common cause of deathamong women between 40 and 55 years of age (Forrest, 1990). Theincidence of breast cancer is increasing, especially in older women, butthe cause of this increase is unknown. Malignant melanoma is anotherform of cancer whose incidence is increasing at a frightening rate, atleast sixfold in the United States since 1945, and is the single mostdeadly of all skin diseases (Fitzpatrick, 1986).

One of the devastating aspects of cancer is the propensity of cells frommalignant neoplasms which disseminate from their primary site to distantorgans and develop into metastatic cancers. Animal tests indicate thatabout 0.01% of circulating cancer cells from solid tumors establishsuccessful metastatic colonies (Fidler, 1993). Despite advances insurgical treatment of primary neoplasms and aggressive therapies, mostcancer patients die as a result of metastatic disease. Hence, there is aneed for new and more efficacious cures for cancer.

The ends of chromosomes have specialized sequences, termed telomeres,comprising tandem repeats of simple DNA sequences. Human telomeresconsist of the sequence 5′-TTAGGG (SEQ ID No. 1) (Blackburn, 1991;Blackburn et al., 1995). Telomeres have several functions apart fromprotecting the ends of chromosomes, the most important of which appearto be associated with senescence, replication, and the cell cycle clock(Counter et al., 1992). Progressive rounds of cell division result in ashortening of the telomeres by some 50-200 nucleotides per round. Almostall tumor cells have shortened telomeres, which are maintained at aconstant length (Allshire et al., 1988; Harley et al., 1990; Harley etal., 1994) and are associated with chromosome instability and cellimmortalization.

The enzyme telomerase adds the telomeric repeat sequences onto telomereends, ensuring the net maintenance of telomere length in tumor cellscommensurate with successive rounds of cell division. Telomerase is aDNA polymerase with an endogenous RNA template (Feng et al., 1995), onwhich the nascent telomeric repeats are synthesized. A significantrecent finding has been that approximately 85-90% of all human cancersare positive for telomerase, both in cultured tumor cells and primarytumor tissue, whereas most somatic cells appear to lack detectablelevels of telomerase (Kim et al., 1994; Hiyama et al., 1995a). Thisfinding has been extended to a wide range of human tumors (see, forexample, references Broccoli, 1994 and Hiyama et al., 1995b) and islikely to be of use in diagnosis.

Human telomerase has been proposed as a novel and potentially highlyselective target for antitumor drug design (Feng et al., 1995; Rhyu etal., 1995; Parkinson, 1996). Studies with antisense constructs againsttelomerase RNA in HeLa cells show that telomere shortening is produced,together with the death of these otherwise immortal cells (Feng et al.,1995). Sequence-specific peptide-nucleic acids directed againsttelomerase RNA have also been found to exert an inhibitory effect on theenzyme Norton et al, 1996).

Among chemical agents, 2,6-diamido-anthraquinones have been reported asDNA-interactive agents (Collier and Neidle, 1988; 1992; Agbandje et al.,1992). These compounds have been shown to act as selective DNA triplexinteractive compounds (Fox et al., 1995; Haq et al., 1996), with reducedaffinity for duplex DNA and only moderate conventional cytotoxicity in arange of tumor cell lines. A carbocyanine dye,3,3′-diethyloxadicarbocyanine (DODC,), has been reported to bind dimerichairpin G-quadruplex structures (Chen et al., 1996).

This invention describes a novel class of non-nucleoside molecules thatare telomerase inhibitors. These compounds have demonstrated theirability to interact with telomeres which form structures called theG-quadruplex structures. As telomeres are involved in controlling thecell cycle, cell replication and aging, these inhibitors of telomeraseprevent uncontrolled cell growth and the immortality of tumor cells.

SUMMARY OF THE INVENTION

The present invention has demonstrated for the first time that anon-nucleoside, small molecule can target the G-quadruplex structure andcan act as a telomerase inhibitor. Accordingly, methods have beendeveloped that identify these classes of compounds and severalinhibitors identified.

Compounds such as those described here, which interact selectively withG-quadruplex structures and inhibit telomerase, are expected to beuseful as inhibitors of the proliferation of cells that requiretelomerase to maintain telomere length for continued growth. Theinvention thus relates to novel methods for identifying compounds thatwill be useful in this regard, and also includes new classes oftelomerase inhibitors. In this regard, several perylene compounds andcarbocyanines have been shown to interact with G-quadruplex structures.Since telomerase appears to be found almost exclusively in tumor cells,these agents are contemplated to be useful as antitumor agents.

In one aspect of the invention, compounds that act as telomeraseinhibitors have been identified. It has been found that compounds thatbind to the human G-quadruplex structure inhibit the human telomerase.The identification of such G-quadruplex interactive agents is anefficient approach for identifying human telomerase inhibitors. Methodsfor identifying these G-quadruplex interactive agents includeidentifying compounds whose three-dimensional structure is complementaryto that of the G-quadruplex structure. Another method for identifyingG-quadruplex interactive compounds is to identify compounds thatinteract with G-quadruplexes using such methods as dye displacement ormelting points of G-quadruplex/compound hybrids.

More particularly, candidate compounds that inhibit telomerase activityare identified by first obtaining the three-dimensional structure of acompound that might interact with the G-quadruplex.selected compound.The complementarity of the compound to human telomere DNA G-quadruplexis then determined. If there is a high degree of complementarity,telomerase inhibition activity is indicated.

Alternatively, one can contact a telomerase inhibitor candidate compoundwith human DNA G-quadruplex; and then determine the melting point of thehuman DNA G-quadruplex. The inventors have found that an increase inmelting point of the quadruplex indicates telomerase inhibitory activityof the compound.

Additionally, telomerase inhibitors may be identified by first preparinga DNA G-quadruplex/dye complex with a dye intercalated into theG-quadruplex; then contacting complex with a telomerase inhibitorcandidate. Displacement of the dye in the complex identifies thecandidate as a telomerase inhibitor.

Yet another aspect of this invention is to provide non-nucleosideinhibitors of telomerase. Using the disclosed screening methods,compounds have been identified that bind to human G-quadruplexstructures. The invention includes perylene compounds, exemplified byN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide that are useful telomerase inhibitors. Novel compounds such asN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimideare also within the scope of the invention.

A preferred G-quadruplex structure is formed from the sequenced(AGGGTTAGGGTTAGGGTTAGGG) (SEQ ID No. 2) or the sequences d(TTAGGG)₄(SEQ ID No. 1), d(TAAGGGT)₄ (SEQ ID No. 3), or d(TTAGGGTT)₄ (SEQ ID No.4) either alone or in the presence of a G-quadruplex interactiveperylene diimide of general structure I. The structures were determinedby NMR spectroscopy. Alternatively, one may determine thethree-dimensional structure of potential G-quadruplex interactive agentsby x-ray diffraction or molecular mechanics calculations. Preferredprograms for determining the degree of complementarity between thepotential G-quadruplex interactive agent and these G-quadruplexstructures include DOCK, autoDOCK, AMBER and SYBYL. The preferredmethods for generating orientations between the potential G-quadruplexinteractive agents and these G-quadruplex structures are manual andusing the DOCK or autoDOCK programs. The cutoff for determining thelikelihood that the orientation of the potential G-quadruplexinteractive agent and the G-quadruplex structure have sufficientchemical interaction to form a complex is roughly 75% of the favorableintermolecular interaction energy, calculated with the above programs,of the perylene diimide 2-d(TTAGGG)₄ (SEQ ID No. 1) complex structure asdetermined by NMR spectroscopy.

Preferred G-quadruplex structures are those formed by the sequencesd(TTAGGG)₄ (SEQ ID No. 1), d(AATGGGT)₄ (SEQ ID No. 5) and d(TTAGGGTT)₄(SEQ ID No. 4). Several methods of determining the interaction ofpotential G-quadruplex interactive agents with these structures includeUV/VIS spectroscopy, in which the changes in the UV/VIS spectrum of thepotential agent under more than a 10% change at the wavelength duesolely to the ligand and which undergoes the most change, upon additionof an excess of the G-quadruplex structure; UV spectroscopy, in whichthe melting temperature of the G-quadruplex structure as determined by ahyperchromicity transition at a given temperature range is increasedby >50° C. upon addition of an excess of the agent; UV/VIS spectroscopyin which addition of a potential G-quadruplex interactive agent to acomplex of a G-quadruplex-interactive perylene diimide and aG-quadruplex produces a >25% change in the absorption of due to theG-quadruplex-interactive perylene diimine-G-quadruplex complex; UV/VISspectroscopy in which addition of a potential G-quadruplex interactiveagent to a complex of a G-quadruplex-interactive carbocyanine and aG-quadruplex produces a >25% change in the absorption of due to theG-quadruplex-interactive carbocyanine-G-quadruplex complex; NMRspectroscopy in which the melting temperature of the G-quadruplex asdetermined by the disappearance of the imino proton signals of theG-quadruplex is increase by >5° C. in the presence of one- totwo-equivalents of the agent; NMR spectroscopy in which the interactionof the agent with the G-quadruplex structure is determined by the shiftof at least one of the imino protons of the G-quadruplex by >0.01 ppmupon addition of one- to two-equivalents of the agent; fluorescencespectroscopy in which the fluorescence emission spectrum of the agentundergoes a shift of >5 nm and/or a change in intensity of >25% upon theaddition of an excess of the G-quadruplex structure; fluorescencespectroscopy in which the fluorescence emission spectrum of aG-quadruplex-interactive perylene diimide-G-quadruplex complex undergoesa >25% change upon the addition of an excess of the agent; orfluorescence spectroscopy in which the fluorescence emission spectrum ofa G-quadruplex-interactive carbocyanine-G-quadruplex complex undergoesa >25% change upon the addition of an excess of the agent.

The preferred embodiments of the invention as it related to one class ofG-quadruplex interactive telomerase inhibitors are compounds of thestructure I in which

in which R¹ and R⁴ are independently taken from the set ofsub-structures given by the formula -L-A in which L is a linking grouptaken from the set consisting of:

where n is 1, 2, or 3; and each R5 is independently taken from the setH, Me, OH, or OMe;

where R5 is as before and Y is taken from the set O, S, SO, SO2, NH,NMe, NCOMe;

where R5 and Y are as before and X is taken from the set CH2, O, S, SO,SO2, NH, NMe, NCOMe;

where R6, R7, R8, and R9 are independently taken from the set consistingof H, OMe, OEt, halo, or Me;

or a bond;

and A is taken from the set consisting of:

where m is 0-5 and each R6 is taken from the set consisting of halo,NH2, NO2, CN, OMe, SO2NH2, amidino, guanidino, or Me;

where o is 0 or 1; p is 0, 1, or 2; q is 1 or 2 such that o+q is either2, in which case a pyrrolidine or pyrrole ring is indicated, or 3, inwhich a piperidine or pyridine ring is indicated; r is 0, 1, 2, or 3; R7is H or Me; each R8 is independently taken from the set consisting ofMe, NO2, OH, CH2OH, halo, or when r is 2 or 3, two adjacent R8substituents may be together taken as —(CH═CH)2— or —(CH2)4— such as toform an annulated six-membered ring;

where each R9 is independently taken from the set consisting of H, Me,or both R9 can taken together be ═O; s is equal to 0 or 1; and Z istaken from the set consisting of CH2, O, NH, NMe, NEt, N(Me)2, N(Et)2,or NCO2Et;

where Q is either N, CH, NMe, or NEt; X is either O, S, NH, NMe or NEt;R10 and R11 are independently taken from the set consisting of H, Me,CH2CO2Et, or R10 and R11 taken together consist of —(CH═CH)2— or—(CH2)4—;

where t is equal to 1, 2, 3, or 4; u is equal to 0, 1, 2, 3, or 4, andeach R12 is individually taken from the set consisting of Me, or OH;

OH, CO2R¹³, CON(R¹³)₂, SO3H, SO2N(R¹³)₂, CN, CH(CO2R¹³)₂,CH(CON(R¹³)₂)₂, N(R¹³)₂, or N(R¹³)₃ where R13 is either H, Me, Et, orCH₂CH₂OH;

R2, R2′, R2″, R2″; R3, R3′, R3″, R3′″ are each independently taken fromthe set H, OMe, halo, or NO2.

In addition, this invention includes the development of otherG-quadruplex interactive telomerase inhibitors compounds derived fromstructure I, having the following structures:

The preferred embodiment of the invention as it relates to another classof G-quadruplex interactive telomerase inhibitors are compounds of thegeneral structure II:

in which C is either a bond, —CH═CH—, —(CH═CH)2—, —(CH═CH)3—,p-phenylene, o-phenylene, p-phenylene-CH═CH—, or o-phenylene-CH═CH—; Bis O, S, or NR, and R is either Me or Et.

In addition, this invention includes the development of anotherG-quadruplex interactive telomerase inhibitor compound derived fromstructure H, having the following structure:

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Effect of increasing concentrations ofN,N′-bis(2-dimethylaminoethyl)-3,4,9, 10-perylenetetracarboxylic aciddiimide on inhibition of telomerase catalyzed extension of an 18-merprimer d[TTAGGG]₃ (SEQ ID No. 1) (1 μM). Elongated primer was labeledwith 1.5 μM of [α-³²P]-dGTP (800 Ci mmol⁻¹, 10 mCi ml⁻¹) with 1 mM dATPand dTTP using a standard telomerase assay. Lanes 1-5 are 0, 10, 50, and100 μM ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide.

FIG. 2. Changes in the UV/VIS absorbance spectrum ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide upon addition of increasing amount of [d(TTAGGGT)]₄ (SEQ ID No.6), an oligodeoxyribonucleotide which adopts a G-quadruplex structure.

FIG. 3. Titration of [d(TTAGGGy]₄ (SEQ ID No. 1) withN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. Imino proton region of the 500-MHz 1H NMR is shown withincreasing amounts of added ligand. The resonances labeled G4*, G5*, andG6* represent resonances of final 2:1 ligand/G-quadruplex complexes.

FIG. 4. NMR-based model of [d(TTAGGG)]₄ (SEQ ID No. 1)-N,N′-bis(2-piperdinoethyl)3,4,9,10-perylenetetracarboxylic acid diimidecomplex. The ligand is stacked under the G6 guanine tetrad withpositively charged side chains located in the grooves.

FIG. 5. Shows the design of an intramolecular quadruplex DNA thatcontains the human telomere repeats.

FIG. 6. Photocleavage of G4A DNA by TMPyP4 in K⁺ buffer.

FIG. 7. Model depicting G-quadruplex structure blocking primer extensionby DNA polymerase.

FIG. 8. Primer extension of PQ sequence in the presence of compounds.Lane 1: water control; Lane 2: 50 mM K+; Lanes 3-6; QQ23; Lanes 7-10;QQ30; Lanes 11-14; QQ31; Lanes 15-18; APPER(Bis[1-(2-aminoethyl)piperdine]-3, 4, 9, 10-perylenetetracarboxylicdiimide); Lanes 3, 7, 11, and 15; 0.5 μM; Lanes 4, 6, 12,and 16; 1 μM;Lanes 5, 9, 13 and 17; 10 μM; Lanes 6, 10, 14 and 18; 50 μM.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS I. The Present Invention

A structure-based approach to discovering non-nucleoside compounds thatwill selectively inhibit human telomerase by targeting the nucleic acidstructures, such as G-quadruplexes, that may be associated with humantelomeres or telomerase has been utilized. Inhibition of humantelomerase by a 2,6-diamido anthraquinone has been successfullydemonstrated. ¹H-NMR has demonstrated the stabilization of aG-quadruplex by this compound and evidence has been provided that thiscompound inhibits the telomerase enzyme by a mechanism consistent withinteraction with G-quadruplex structures. The present work shows thatnon-nucleoside, small molecules can interact with G-quadruplexes andinhibit telomerase.

Using the methods described, it was found that compounds that bind tothe human G-quadruplex structure inhibit the human telomerase. Theidentification of such G-quadruplex interactive agents is a novel andefficient approach for identifying human telomerase inhibitors.

It is envisioned that the telomerase inhibitors will provide therapy fortumors and cancers including skin cancers, connective tissue cancers,adipose cancers, breast cancers, lung cancers, stomach cancers,pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers,anogenital cancers, kidney cancers, bladder cancers, colon cancers,prostate cancers, central nervous system (CNS) cancers, retinal cancer,blood, lymphoid cancers and the like.

II. Telomerase

Telomerase is a ribonucleoprotein enzyme that synthesizes one strand ofthe telomeric DNA using as a template a sequence contained within theRNA component of the enzyme. The ends of chromosomes have specializedsequences, termed telomeres, comprising tandem repeats of simple DNAsequences which in humans is 5′-TTAGGG (SEQ ID No. 1) (Blackburn, 1991;Blackburn et al., 1995). Apart from protecting ends of chromosomestelomeres have several other functions, the most important of whichappear to be associated with replication, regulating the cell cycleclock and ageing (Counter et al., 1992). Progressive rounds of celldivision shorten telomeres by 50-200 nucleotides per round. Almost alltumor cells have shortened telomeres, which are maintained at a constantlength (Allshire et al., 1988; Harley et al., 1990; Harley et al., 1994)and are associated with chromosome instability and cell immortalization.

With regard to human cells and tissues telomerase activity has beenidentified in immortal cell lines and in ovarian carcinoma but has notbeen detected at biologically significant levels (that are required tomaintain telomere length over many cell divisions) in mortal cellstrains or in normal non-germline tissues (Counter et al., 1992; Counteret al, 1994). These observations suggest telomerase activity is directlyinvolved in telomere maintenance, linking this enzyme to cellimmortality.

As described above, the immortalization of cells involves the activationof telomerase. More specifically, the connection between telomeraseactivity and the ability of many tumor cell lines, including skin,connective tissue, adipose, breast, lung, stomach, pancreas, ovary,cervix, uterus, kidney, bladder, colon, prostate, central nervous system(CNS), retina and blood tumor cell lines, to remain immortal has beendemonstrated by analysis of telomerase activity (Kim, et al., 1994).This analysis, supplemented by data that indicates that the shorteningof telomere length can provide the signal for replicative senescence innormal cells, see PCT Application No. 93/23572, incorporated herein byreference, demonstrates that inhibition of telomerase activity can be aneffective anti-cancer therapy. Thus, telomerase activity can prevent theonset of otherwise normal replicative senescence by preventing thenormal reduction of telomere length and the concurrent cessation of cellreplication that occurs in normal somatic cells after many celldivisions. In cancer cells, where the malignant phenotype is due to lossof cell cycle or growth controls or other genetic damage, an absence oftelomerase activity permits the loss of telomeric DNA during celldivision, resulting in chromosomal rearrangements and aberrations thatlead ultimately to cell death. However, in cancer cells havingtelomerase activity, telomeric DNA is not lost during cell division,thereby allowing the cancer cells to become immortal, leading to aterminal prognosis for the patient.

Methods for detecting telomerase activity, as well as for identifyingcompounds that regulate or affect telomerase activity, together withmethods for therapy and diagnosis of cellular senescence andimmortalization by controlling telomere length and telomerase activity,have also been described elsewhere.

A. G-Quadruplex Structures

Human telomeres form structures known as G-quadruplexes. Human telomerescontain numerous repeats of the sequence TTAGGG (SEQ ID No. 1),exhibiting an enhancement of G and T residues and a paucity of Aresidues. Intramolecular G-quadruplex DNA may be designed by generatinga sequence of human telomere repeats (FIG. 5). The G tetrad consists offour G bases hydrogen bonded in Hoogsteen fashion symmetrically disposedabout a central axis, as shown in FIG. 5.

G-rich DNA is known to assume highly stable structures formed byHoogsteen base pairs between guanine residues (Williamson, 1994; Nadelet at., 1995). These structures, known as G-quadruplexes, are stabilizedin the presence of K⁻and may have biological roles that are yet to bedetermined (Henderson et at., 1987; Hardin et at., 1997; Williamson etat., 1989). One particular region of the genome where these structuresmay play a significant biological role is at the ends of chromosomeswhere G-rich DNA is normally found (e.g., TTAGGG (SEQ ID No. 1) andTTGGGG (SEQ ID No. 7) tandem repeats in human cells and ciliateTetrahymena, respectively) (Henderson et at., 1987; Blackburn andGreider, 1995; Sundquist and Heaphy, 1993). In addition, a number ofgenes containing G-rich DNA have been identified recently, and it hasbeen proposed that the G-rich regions within these genes may regulategene expression by forming G-quadruplex structures (Sen and Gilbert,1988; Hommond-Kosack et at., 1993; Murchie and Lilley, 1992; Simonssonet al., 1998). One potential biologically relevant role of G-quadruplexDNA is as a barrier to DNA synthesis (Howell et al., 1996). This barrierhas been thoroughly investigated and has been found to be K⁺dependent(Woodword et at., 1994). This observation strongly suggests that theformation of G-quadruplex species is responsible for the observed effecton DNA synthesis (Weitzmann et al., 1996).

The inventors have shown that the 2,6-diamidoanthraquinone BSU-1051modulates human telomerase activity by a mechanism that is dependent onthe elongation of the telomeric primer d(TTAGGG)₃ (SEQ ID No. 1) to alength that is then capable of forming an intramolecular G-quadruplexstructure (Sun et al., 1997). The inventors have also shown thatBSU-1051, by virtue of its interaction with G-quadruplex DNA, enhancesthe block of DNA synthesis by the G-quadruplex structure in the presenceof K⁺.

B. Methods for Identifying G-quadruplex Interactive Agents

Several methods for identifying classes of G-quadruplex interactiveagents may be employed. One method involves identifying compounds whosethree-dimensional structure is complementary to that of the G-quadruplexstructure. G-quadruplex structure is understood to mean at least in onesense the structure of the G-quadruplex that is formed by thesingle-stranded DNA corresponding to at least four repeats of thetelomeric sequence. In humans, the telomeric sequence is d(TTAGGG) (SEQID No. 1). Thus, the G-quadruplex structure of interest for theidentification of human telomerase inhibitors may be any sequence of theform {d([N₁]TTAGGG[N₂])}₄ (SEQ ID No. 1) where [N₁] is zero to two basescorresponding to the human telomeric sequence; for example, [N₁] mayequal G, GG, or may be absent; where [N₂] is zero to three basescorresponding to the human telomeric sequence; for example, [N₂] canequal T, TT, TTA or it may be absent.

Alternatively, G-quadruplex structure is understood to mean thefold-over or intramolecular G-quadruplex formed from at least fourrepeats of the G-triad of telomeric sequence. Thus, the G-quadruplexstructure of interest for the identification of human telomeraseinhibitors may be any sequence of the form d([N₃][TTAGGG]₃[N₂]) (SEQ IDNo. 1 where [N₂] is as defined above and [N₃] is three G's preceded byzero to three nucleotides corresponding to the human telomeric sequence.These structures may be determined by a variety of techniques includingmolecular mechanics calculations, molecular dynamics calculations,constrained molecular dynamics calculations in which the constraints aredetermined by NMR spectroscopy, distance geometry in which the distancematrix is partially determined by NMR spectroscopy, x-ray diffraction,or neutron diffraction techniques. In the case of all these techniques,the structure can be determined in the presence or absence of anyligands known to interact with G-quadruplex structures, including butnot limited to potassium and other metal ions,2,6-diamidoanthraquinones, perylene diimides, or carbocyanines.

Complementary is understood to mean the existence of a chemicalattraction between the G-quadruplex interactive agent and theG-quadruplex. The chemical interaction may be due to one or a variety offavorable interactions, including ionic, ion-dipole, dipole—dipole, vander Waals, charge-transfer, and hydrophobic interactions. Each of thesetype of interactions, alone or together, may be determined by existingcomputer programs using as inputs the structure of the compound, thestructure of the G-quadruplex, and the relative orientation of the two.Such computer programs include but are not limited to AMBER, CHARMM,MM2, SYBYL, CHEMX, MACROMODEL, GRID, and BioSym. Such programs arecontemplated as being useful for the determination of the chemicalinteraction between two molecules, either isolated, or surrounded bysolvent molecules, such as water molecules, or using calculationaltechniques that approximate the effect of solvating the interactingmolecules. The relative orientation of the two can be determinedmanually, by visual inspection, or by using other computer programswhich generate a large number of possible orientations.

Examples of computer programs include but are not limited to DOCK andAutoDOCK. Each orientation can be tested for its degree ofcomplementarity using the computer programs. An advantage of this methodis that it does not require availability of physical samples of thecompounds, only that their three-dimensional structure is known. It thuscan be used to design novel compounds that possess the desired abilityto inhibit telomerase.

Alternatively, this method may be used as a screening method foridentifying telomerase inhibitors from a collection of compounds thatare available, provided that the structure of these compounds is known.If only the two-dimensional structure is known, the correspondingthree-dimensional structure can be obtained using existing computerprograms. Such computer programs include but are not limited to CONCORD,CHEM3D, and MM2.

Another method for identifying G-quadruplex interactive compounds thatmay inhibit telomerase involves use of techniques such as UV/VISspectroscopy, polarimetry, CD or ORD spectroscopy, IR or Ramanspectroscopy, NMR spectroscopy, fluorescence spectroscopy, HPLC, gelelectrophoresis, capillary gel electrophoresis, dialysis, refractometry,conductometry, atomic force microscopy, polarography, dielectometry,calorimetry, solubility, EPR or mass spectroscopy. The application ofthese methods can be direct, in which the G-quadruplex interactivecompound's interaction with the G-quadruplex is measured directly, or itcan be indirect, in which a particular G-quadruplex interactive agenthaving a useful spectroscopic property is used as a probe for theability of other compounds to bind to the G-quadruplex; for example, bydisplacement or by fluorescence quenching.

III. Telomerase Inhibitors

The identification of compounds that inhibit telomerase activityprovides important benefits to efforts at treating human disease.Compounds that inhibit telomerase activity can be used to treat cancer,as cancer cells express telomerase activity and normal human somaticcells do not express telomerase activity at biologically relevant levels(i.e. at levels sufficient to maintain telomere length over many celldivisions). Unfortunately, few such compounds have been identified andcharacterized. Hence, there remains a need for compounds that act astelomerase inhibitors and for compositions and methods for treatingcancer and other diseases in telomerase activity is present abnormally.The present invention meets these and other needs.

Once a compound has been identified as being a G-quadruplex interactiveagent, confirmatory evidence for the ability of said compound to inhibittelomerase may be obtained using a standard primer extension assay thatdoes not use a PCR™-based amplification of the telomerase primerextension products such as described in Sun et al., 1997. The identifiedinhibitors may be used therapeutically to interfere with the function oftelomerase and thus to treat cancers.

Using the screening methods described above, compounds have beenidentified that bind to human G-quadruplex structures and have beenshown to inhibit human telomerase. One group of compounds is representedby general structure I:

in which R¹ and R⁴ are represented by L-A where L is a linking groupwhich may be any of a group of substituted (X)methylene, (X)dimethylene,(X)trimethylene, (X)dimethyleneamine, (X)dimethyleneoxy,(X)dimethyleneaminodimethylene, (X)-dimethyleneoxydimethylene,(X)-p-phenylene, (X)-m-phenylene, (X)-o-phenylene, or an unsubstitutedcovalent bond;

A is a group that interacts with the grooves of the G-quadruplexstructure, examples being a substituted carbocyclic ring, a(substituted) heterocyclic ring, an hydroxyl, a carboxylic acid, acarboxylic acid ester, a carboxamide, a sulfonamide, a sulfonic acid, anitrile, a malonate diester, a malonate diamide, a disubstituted amine,a quarternized nitrogen-containing heterocyclce, or a quaternary amine.

R2, R2′, R2″, R2′″, R3, R3′, R3″, R3′″ are independently hydrogen,alkyl, halo, amino, nitro, hydroxy, alkoxy, alkylamino, dialkylamino,aryl, or cyano.

Another group of compounds suitable as telomerase inhibitors is shown bythe general structure II in which B is O, S, or NR; C is an unsaturatedlinking group, 1 to 3 (substituted) ethylene groups, a substituted orunsubstituted carbocyclic group, or a heterocyclic group; and R is loweralkyl.

These compounds may interact specifically with G-quadruplex structuresas compared to other nucleic acid structures such as double-strandedDNA, single-stranded DNA, and RNA structures. The degree of selectivityin the interaction of these compounds with G-quadruplex structuresversus other nucleic acid structures is given by the ratio of theaffinity of these compounds for G-qudruplex structures to the affinityfor the other nucleic acid structures. In particular, the ability ofthese compounds to distinguish between G-quadruplex structures anddouble-stranded DNA may be an important criterion. Compounds withgeneral ability to bind double-stranded DNA are known to inhibit oralter a variety of DNA-associated enzymes or proteins, including but notlimited to: histone binding, topoisomerase I, topoisomerase II,DNA-polymerases, RNA-polymerases, DNA repair enzymes, cytosinemethyltransferase, and transcription factor binding. In order todiscover compounds that are able to selectively inhibit telomerase andother G-qudruplex-associated enzymes and proteins, one would want toselect compounds that have a high ratio of affinities for G-qudruplexstructures versus double-stranded DNA. These selectiveG-quadruplex-binding compounds can be identified by selecting thoseG-quadruplex binding compounds that display weak or no ability forbinding to double-stranded DNA, as determined by UV/VIS spectroscopy,polarimetry, CD or ORD spectroscopy, IR or Raman spectroscopy, NMRspectroscopy, fluorescence spectroscopy, HPLC, gel electrophoresis,capillary gel electrophoresis, dialysis, refractometry, conductometry,atomic force microscopy, polarography, dielectometry, calorimetry,solubility, EPR and mass spectroscopy.

In addition to the thermodynamic considerations of G-quadruplex bindingby these compounds, the kinetics of the interaction between thesecompounds and G-quadruplexes is also considered to be important. Therelative rates of the association and dissociation of these compoundswith G-quadruplex structures can affect their biological properties. Inparticular, those compounds with slow dissociation rates may be moreeffective in inhibiting telomerase and other G-quadruplex-associatedenzymes and proteins than hose with identical G-quadruplex bindingability, but whose dissociation rates are faster. For a given compound,the overall equilibrium binding affinity (binding constant) toG-quadruplex structures is a ratio of the association rate and thedissociation rate. The dissociation rate of a complex consisting of aG-quadruplex structure and a G-quadruplex interactive compound can bedetermined by a variety of methods. In one example, the dissociation ofthe complex can be determined spectrophotometrically upon the additionof a detergent, such as SDS. Alternatively, the dissociation rate can bedetermined in a T-jump study, in which the temperature of the complex isquickly raised to a point at which the complex dissociates and thisprocess is monitored spectrophotometrically. In another example thedissociation rate for a G-quadruplex-compound complex can be determinedby monitoring by a variety of techniques the dissociation of a complexin which one partner, either the G-quadruplex or the compound, istethered, either covalently or non-covalently, to an immobile phase, anda solution is passed over this immobilized complex. The dissociationrate for a G-quadruplex-compound complex also can be determinedindirectly, by measuring both the equilibrium binding constant and theassociation rate. For example, if two compounds have similar equilibriumG-quadruplex binding constants, but one has a slower association rate,then that same compound must also have a proportionately slowdissociation rate.

Using the above techniques, one can select from those G-quadruplexinteractive agents identified, those that have additional desiredproperties of selective G-quadruplex interaction when compared to othernucleic acids structures, such as double-stranded DNA, and/or slowkinetics of association with G-quadruplex structures.

Agents capable of inhibiting telomerase activity in tumor cells offertherapeutic benefits with respect to a wide variety of cancers and otherconditions (for example, fungal infections) in which immortalized cellstelomerase activity are a factor in disease progression or in whichinhibition of telomerase activity is desired for treatment purposes. Thetelomerase inhibitors of the invention can also be used to inhibittelomerase activity in germ line cells, which may be useful forcontraceptive purposes.

IV. Pharmaceutical Formulations and Administration

The invention further comprises the therapeutic treatment of cancer bythe administration of an effective dose of one or more inhibitors oftelomerase. Where clinical applications are contemplated, it will benecessary to prepare pharmaceutical compositions of drugs in a formappropriate for the intended application. Generally, this will entailpreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to humans or animals.

The phrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well know in the art. Supplementary active ingredients also can beincorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra. A preferred route is direct intra-tumoral injection,injection into the tumor vasculature or local or regional administrationrelative to the tumor site.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the compounds developed in the present inventionmay be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Because telomerase is active only in tumor, germline, and certain stemcells of the hematopoietic system, other normal cells are not affectedby telomerase inhibition therapy Steps also can be taken to avoidcontact of telomerase inhibitor with germline or stem cells, althoughthis may not be essential. For instance, because germline cells expresstelomerase activity, inhibition telomerase may negatively impactspermatogenesis and sperm viability, suggesting that telomeraseinhibitors may be effective contraceptives or sterilization agents. Thiscontraceptive effect may not be desired, however, by a patient receivinga telomerase inhibitor of the invention for treatment of cancer. In suchcases, one can deliver a telomerase inhibitor of the invention in amanner that ensures the inhibitor will only be produced during theperiod of therapy, such that the negative impact on germline cells isonly transient.

V. Therapies

One of the major challenges in oncology today is the effective treatmentof a given tumor. Tumors are often resistant to traditional therapies.Thus, a great deal of effort is being directed at finding efficoustreatment of cancer. One way of achieving this is by combining new drugswith the traditional therapies and is discussed below. In the context ofthe present invention, it is contemplated that therapies directedagainst telomerase could be used in conjunction with surgery,chemotherapy, radiotherapy and indeed gene therapeutic intervention. Italso may prove effective to combine telomerase targeted chemotherapywith antisense or immunotherapies directed toward tumor markers or otheroncogenes or oncoproteins.

“Effective amounts” are those amounts of a candidate substance effectiveto reproducibly decrease expression of telomerase in an assay incomparison to levels in untreated cells. An “effective amount” also isdefined as an amount that will decrease, reduce, inhibit or otherwiseabrogate the growth of a cancer cell.

It is envisioned that the telomerase inhibitors will provide therapy fora wide variety of tumors and cancers including skin cancers, connectivetissue cancers, adipose cancers, breast cancers, lung cancers, stomachcancers, pancreatic cancers, ovarian cancers, cervical cancers, uterinecancers, anogenital cancers, kidney cancers, bladder cancers, coloncancers, prostate cancers, central nervous system (CNS) cancers, retinalcancer, blood and lymphoid cancers.

A. Combination Therapies

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, the methods of standard therapy discussed above aregenerally insufficient as tumors are often resistant to several of theseagents. Often combining a host of different treatment methods prove mosteffective in cancer therapy. Further, several AIDS afflicted patientshave a higher risk of developing cancers. Combination therapy in thesecases is required to treat AIDS as well as the cancer. Using the methodsand compounds developed in the present invention, one would generallycontact a “target” cell with a telomerase inhibitor and at least oneother agent. These compositions would be provided in a combined amounteffective to kill or inhibit proliferation of the cell. This process mayinvolve contacting the cells with the telomerase based therapy and theother agent(s) or factor(s) at the same time. This may also be achievedby contacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the telomerase based therapy and the other includesthe agent.

Alternatively, the telomerase inhibitor-based treatment may precede orfollow the other agent treatment by intervals ranging from min to wk. Inembodiments where the other agent and telomerase-based therapy areapplied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and telomerase-based treatment would stillbe able to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other, with a delay time of only about 12 hbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of eithertelomerase-based treatment or the other agent will be desired. Variouscombinations may be employed, where telomerase-based treatment is “A”and the other agent is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are also contemplated. Again, to achieve cellkilling, both agents are delivered to a cell in a combined amounteffective to kill the cell.

The invention also encompasses the use of a combination of one or moreDNA damaging agents, whether chemotherapeutic compounds orradiotherapeutics as described in the section below, together with thetelomerase inhibitor. The invention also contemplates the use of thetelomerase inhibitors in combination with surgical removal of tumors totreat any remaining neoplastic or metastasized cells. Further,immunotherapy may be directed at tumor antigen markers that are found onthe surface of tumor cells. The invention also contemplates the use oftelomerase inhibitors in combination with gene therapy, directed towarda variety of oncogenes, such as, tumor markers, cell cycle controllinggenes, described below.

The other agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with the telomerase-inhibitor basedtreatment, as described above. The skilled artisan is directed to“Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, inparticular pages 624-652. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

It is proposed that the regional delivery of non-nucleoside inhibitorsof telomerase to patients with tumors will be a very efficient methodfor delivering a therapeutically effective chemical to counteract theclinical disease. Similarly, other chemotherapeutics, radiotherapeutics,gene therapeutic agents may be directed to a particular, affected regionof the subjects body. Alternatively, systemic delivery of telomerasebased treatment and/or the agent may be appropriate in certaincircumstances, for example, where extensive metastasis has occurred.

It also should be pointed out that any of the standard or othertherapies may prove useful by themselves in treating a cancer. In thisregard, reference to chemotherapeutics and non-telomeraseinhibitor-based treatment in combination should also be read as acontemplation that these approaches may be employed separately.

When such combination therapy is employed for the treatment of a tumor,the cytotoxic agent may be administered at a dosage known in the art tobe effective for treating the tumor. However, the G-quadruplexinteraction compounds may produce an additive or synergistic effect witha cytotoxic agent against a particular tumor. Thus, when suchcombination antitumor therapy is used, the dosage of G-quadruplexinteraction compounds administered may be less than that administeredwhen the cytotoxic agent is used alone. Similarly, for patientsafflicted by AIDS, AZT/protease inhibitors will be used withG-quadruplex interaction compounds, or other herein mentionedtherapeutic agent(s). Again the dosage of G-quadruplex interactioncompounds or other conjunctively utilized agent, may be altered to suitthe AIDS treatment.

Preferably, the patient is treated with G-quadruplex interactioncompounds for about 1 to 14 days, preferably 4 to 14 days, prior to thebeginning of therapy with a cytotoxic agent, and thereafter, on a dailybasis during the course of such therapy. Daily treatment with thetelomerase inhibitor can be continued for a period of, for example, 1 to365 days after the last dose of the cytotoxic agent is administered.This invention encompasses the use of telomerase inhibitors-based cancertherapy for a wide variety of tumors and cancers affecting skin,connective tissues, adipose, breast, lung, stomach, pancreas, ovary,cervix, uterus, kidney, bladder, colon, prostate, anogenital, centralnervous system (CNS), retina and blood and lymph.

B. Standard Therapies

Described herein are the therapies used as standard or traditionalmethods for treatment of cancers. The section on chemotherapy describesthe use of non-nucleoside telomerase inhibitors as chemotherapeuticagents in addition to several other well known chemotherapeutic agents.As detailed in the section above, all the methods described below can beused in combination with the telomerase inhibitors developed in thepresent invention.

a. Surgery: Surgical treatment for removal of the cancerous growth isgenerally a standard procedure for the treatment of tumors and cancers.This attempts to remove the entire cancerous growth. However, surgery isgenerally combined with chemotherapy and/or radiotherapy to ensure thedestruction of any remaining neoplastic or malignant cells.

b. Chemotherapy: A variety of chemical compounds, also described as“chemotherapeutic agents”, function to induce DNA damage, are used totreat tumors. Chemotherapeutic agents contemplated to be of use,include, adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin, cisplatin (CDDP), hydrogenperoxide, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, tamoxifen, taxol,transplatinum, vincristin, vinblastin and methotrexate to mention a few.

Agents that damage DNA include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. A number of such agentshave been developed, particularly useful are agents that have undergoneextensive testing and are readily available. 5-fluorouracil (5-FU), isone such agent that is preferentially used by neoplastic tissue, makingit particularly useful for targeting neoplastic cells. Thus, althoughquite toxic, 5-FU, is applicable with a wide range of carriers,including topical and even intravenous administrations with dosesranging from 3 to 15 mg/kg/day.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a useful antineoplastictreatment. For example, cisplatin, and other DNA alkylating agents maybe used. Cisplatin has been widely used to treat cancer, withefficacious doses used in clinical applications of 20 mg/m² for 5 daysevery three wk for a total of three courses. Cisplatin is not absorbedorally and must therefore be delivered via injection intravenously,subcutaneously, intratumorally or intraperitoneally.

The non-nucleoside G-quadruplex inhibitor compounds developed in thisinvention are chemotherapeutic agents that are cytotoxic and inhibittelomerase function which is critical to cell replication andmaintenance of tumor cell immortality. These compounds also indirectlyinhibit DNA polymerases by their strong interactions with theG-quadruplex structures (FIG. 7).

c. Radiotherapy: Radiotherapeutic agents and factors include radiationand waves that induce DNA damage for example, γ-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions, radioisotopes, and thelike. Therapy may be achieved by irradiating the localized tumor sitewith the above described forms of radiation's. It is most likely thatall of these factors effect a broad range of damage DNA, on theprecursors of DNA, the replication and repair of DNA, and the assemblyand maintenance of chromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 wk), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

d. Gene Therapy: Gene therapy based treatments targeted towardsoncogenes such as p53, p16, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf,erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl, which are oftenmutated versions of their normal cellular counterparts in canceroustissues.

VI. Screening for Anti-telomere and Anti-cancer Activity

In particular embodiments, one may test the inhibitors by measuringtheir ability to inhibit growth of cancer cells, to induce cytotoxicevents in cancer cells, to induce apoptosis of the cancer cells, toreduce tumor burden and to inhibit metastases. For example, one canmeasure cell growth according to the MTT assay. A significant inhibitionin growth is represented by decreases of at least about 30%-40% ascompared to uninhibited, and most preferably, of at least about 50%,with more significant decreases also being possible. Growth assays asmeasured by the MTT assay are well known in the art. Other assays tomeasure cell death, apoptosis are well known in the art, for example,Mosmann et al., 1983; Rubinstein et al., 1990.

Quantitative in vitro testing of the anti-tumor agents identified hereinis not a requirement of the invention as it is generally envisioned thatthe agents will often be selected on the basis of their known propertiesor by structural and/or functional comparison to those agents alreadydemonstrated to be effective. Therefore, the effective amounts willoften be those amounts proposed to be safe for administration to animalsin another context.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Telomerase Inhibition byN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide

Selection ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide as a potential telomerase inhibitor was determined by Method (B)as described in Example 2.

The relative inhibition of telomerase byN,N′-bis(2-dimethylaminoethyl)3,4,9,10-perylenetetracarboxylic aciddiimide was determined in a standard primer extension assay that doesnot use a PCR™-based amplification of the telomerase primer extensionproducts. Briefly, the 18-mer telomeric primer d[TTAGGG]₃ (SEQ ID No. 1)(1 μM) without or with N,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide was elongated with telomerase inthe presence of 1.5 μM of [α-³²P]-dGTP (800 Ci mmol⁻¹, 10 mCi ml⁻¹) with1 mM dATP and 1 mM dTTP. The extension products were isolated andvisualized by autoradiography after denaturing gel electrophoresis.

The IC₅₀ was determined to be 50 μM, and at 100 μM ofN,N′-bis(2-dimethylaminoethyl)3,4,9,10-perylenetetracarboxylic aciddiimide there is an almost complete inhibition of telomerase activity.N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimideand 3 showed similar behavior.

Example 2 Screening Assays for Telomerase Inhibitors

Compounds that inhibit telomerase are potential drugs for the treatmentof cancer. The method selects compounds based upon ability to interactwith the human DNA-G-quadruplex. Several procedures for detecting thisinteraction include:

(A) A three dimensional structure of a candidate compound will beanalyzed to determine their degree of complementarity to thethree-dimensional structure of human telomeric DNA G-quadruplex. The NMRsolution structure of d(AGGGTTAGGGTTAGGGTTAGGG) (SEQ ID No. 8) [pdbentry 143d] and its corresponding molecular surface, generated with thems program, were used as inputs to the SPHGEN program. The resultingsphere cluster was used as input to DOCKv2.0 and a subset of theCambridge crystallographic database was search using the contact scoringalgorithm.N,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide was found to have one of the highest contact scores in the ˜2000compounds examined.

(B) Compounds may be selected for their ability to interact with humanDNA G-quadruplex as indicated by UV/VIS spectroscopy. To a 10 μMsolution ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide in 20 mM phosphate buffer containing 100 mM KCl, pH 7.0 in aquartz cuvette was added 10 μL aliquots of a 3 mM solution ofd(TTAGGGT)₄ (SEQ ID No. 6). After each addition the UV/VIS spectrum wasrecorded. Pronounced changes in the UV/VIS spectrum of the compound werenoted at wavelengths 488 nm (˜40% hypochromicity), 510 nm 50%hyperchromicity), and 548 nm (˜200% hyperchromicity).

(C) Compounds may be selected for their ability to interact with humanDNA G-quadruplex as indicated by NMR spectroscopy. The imino protonspectrum (9-12 ppm) of a solution of d(TTAGGG)4 (SEQ ID No. 1) inD2O/H2O (10:90) was determined at 500 MHz. Aliquot ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimidewere added and the imino proton spectrum recorded. At an overallstoichiometry of 1:1 the G6 imino resonance becomes significantlybroader and shifts >0.2 ppm upfield.

(D) Compounds may be selected for their ability to interact with humanDNA g-quadruplex as indicated by an increase in the melting temperatureof the G-quadruplex structure. Thermal denaturation of the parallelfour-stranded G-quadruplex structure formed by the d[T₂AG₃T] (7-mer)(125 mM KCl, 25 mM KH₂PO₄, 1 mM EDTA, pH 6.9) monitored by NMR. Thespectrum for DNA alone and in the presence ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. The molar ratio ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide to quadruplex was 4:1. The imino proton signals have beenassigned previously (Laughlan et al., 1994) as G6, G5, and G4 from highto low field. The presence of drug leads to line broadening and anupfield shift of the imino proton signals indicative of intercalation.Furthermore, the melting temperature of the DNA G-quadruplex isincreased significantly in the presence of the compound. Spectra wereacquired in 90% H₂O/10% D₂O on a Bruker AMX 500 MHz spectrometer atvarious 15-85° C. using a 1—1 echo pulse sequence with a maximumexcitation centered at 12.0 ppm. A total of 128 scans was obtained foreach spectrum with a relaxation delay of 2 s. Before acquiring thespectrum at each temperature, the sample was allowed to equilibrate atthe new temperature for at least 10 min. The data were processed with anexponential window function using 2 Hz of line broadening. The dataindicate thatN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide increases the melting temperature of the G-quadruplex by atleast 20° C.

Example 3 Synthesis ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide

N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimidewas prepared by mixing three g of 3,4,9,10-perylenetetracarboxylic aciddianhydride with 2.5 mL of 1-(2-aminoethyl)piperidine in 10 mL of DMAand 10 mL of 1,4-dioxane. The mixture was heated under reflux for 6hours, and the solvents removed under reduced pressure. The residue wasdissolved in −100 mL of distilled water, and insoluble components wereremoved by filtration. The pH of the resulting solution was adjusted to−3 with the addition of HCl, and the solution was allowed to standovernight. Precipitated impurities was removed by filtration, and theresulting solution was adjusted to pH 1-12 with the addition of NaOH.The precipitated product was isolated by filtration, washed with waterand dried under vacuum.

Example 4 DNA Synthesis Arrest Assay

It has been shown that DNA sequences with quadruplex-forming potentialpresent obstacles to DNA synthesis by DNA polymerases in a K⁺ dependentmanner. This K⁺ dependent block to DNA polymerase is a selective andsensitive indicator of the formation of intramolecular quadruplexes(Weitzmann, et al., 1996). This assay has been adapted to demonstratethe stabilization of quadruplex by small molecules and used to screenpotential G-quadruplex-interactive compounds.

The assay is a modification of that described by Weitzmann, et al.Briefly, primers (24 nM, sequence: 5′-TAATACGACTCACTATAG-3′) (SEQ ID No.9) labeled with [γ-³²P]ATP were mixed with template DNA PQ74(12 nM,

(SEQ ID No. 10) sequence: TCCAACTATGTATACTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTAGCGGCACGCAATTGCTATAGTGAGTCGTATTA-

ID No. 10) in a Tris-HCl buffer (10 mM Tris, pH 8.0) containing 5 mMK⁺and heated at 90° C. for 4 min. After cooling at room temperature for15 min. potential G-quadruplex-interactive compounds were then added tovarious concentrations. The primer extension reactions were initiated byadding dNTP (final concentration 100 μM), MgCl₂ (final concentration 3mM) and Taq polymerase (2.5 U/reaction, Boehringer Mannheim). Thereactions were incubated at 55° C. for 15 mm. then stopped by adding anequal volume of stop buffer (95% formamide, 10 mM EDTA, 10 mM NaOH, 0.1%xylene cyanol, 0.1% bromophenol blue). The products were separated on a12% polyacrylamide sequencing gel. The gels were then dried andvisualized on a phosphorimager (Molecular Dynamics model 445 S1).

To validate the assay, G-quadruplex interactive compounds such asporphyrins and perylenes were tested. The results were consistent withNMR and telomerase inhibition data. FIG. 8 shows the DNA synthesisarrest induced by Quinobenzoxazine analogs (QQ23, 1130, QQ31) and aperylene compound (APPER).

Example 5 Photocleavage Assay to Detect Quadruplex DNA Interactions (i)Design and Synthesis of an Intramolecular Quadruplex DNA

The oligonucleotide G4A employed was synthesized on a Perseptive DNAsynthesizer and deprotected following the routine phosporamiditeprocedures. the DNA was purified by polyacrylamide gel electrophoresis(PAGE). The sequence for this 39 oligomer single strand DNA is:

5′ CATGGTGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC 3′ (SEQ ID No. 11).

This human telomere repeat-containing DNA was designed to form anintramolecular quadruplex which can be stabilized by the stem region in(FIG. 5). A sticky end was added so that unusual secondary structurescould be detected by ligation assay once they are formed.

(ii) Photocleavage Assay

The G4A DNA was labeled with ³²P at the 5′ end and stored in 1×TE bufferat 3000 cpm/μl. For each photocleavage reaction, 10μ of DNA (˜5 ng) wasmixed with 10 μl of 200 mM KCl or 200 mM NaCl and boiled for 10 minbefore cooled down to room temperature. For the no porphyrin controlsamples, 10 μl of distilled water was added instead. The mixtures weretransferred to a 96 well plate and added with 2 μl of 1 μM TMPyP4aqueous solution. The samples were then exposed to 24 watts fluorescentdaylight under a glass filter for different periods of time. Then thereactions were stopped with 100 μl of calf thymus DNA (0.1 μg/μl). Afterphenol-chloroform extraction, the samples were subjected to strandbreakage treatment and ethanol precipitation. The DNA samples wereloaded onto a 16% polyacrylamide gel for electrophoresis and visualizedwith Phosphorimager (from Molecular Dynamics, Inc.). A typical resultfor the photocleavage assay is shown in FIG. 6.

Example 6 Selection of G-Quadruplex Selective Ligand

The N,N′-bis(3 -morpholinopropyl)3,4,9,10-perylenetetracarboxilic aciddiimide (KeTEL01) was synthesized from 3,4,9,10-perylenetetracarboxylicacid dianhydride and 3-morpholinopropylamine using a procedure analogousto that described above in example 5.3 for the synthesis ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. A solution of KeTEL01 was prepared by dissolving 1 mg ofKeTEL01 in 300 μL of 1 N HCl. To this solution was added 11 mL of a pH7.0 buffer containing 20 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, and0.02% hydroxypropyl-β-cyclodextrin. Aliquots of this stock solution ofKeTEL01 were transferred to 8 different quartz cuvettes and diluted intopH 7.0 mM sodium phosphate, 100 mM KCl, 1 mM EDTA buffer to affordsolutions in which the concentration of KeTEL01 was 20 μM. To each ofthe cuvettes was added a solution of [d(TTAGGGT)]4 (SEQ ID No. 6) sothat the final concentration of [d(TTAGGGT)]4 (SEQ ID No. 6) in each ofthe cuvettes was 0,4, 8, 12, 16, 20, 50, and 80 μM. These solutions wereallowed to stand overnight in the dark, and the UV/VIS spectrum of eachwas determined. Pronounced, G-quadruplex concentration-dependent changesin the UV/VIS spectrum were noted at wavelengths 488 nm (˜40%hypochromicity), 510 nm (˜40% hyperchromicity) and 548 nm (˜100%hyperchromicity). In a parallel study, changes in the UV/VIS spectrum ofa 20 μM solution of KeTEL01 in a pH 7.0 20 mM phosphate buffercontaining 100 mM KCl and 1 mM EDTA were determined upon the addition of10 μM aliquots of a 3 mM (base pair) solution of calf thymus DNA. Nochanges in the UV/VIS spectrum of this solution were noted, indicatingthat KeTEL01 does not interact with double-stranded DNA.

Example 7 Kinetics of Interaction of KeTEL01 with G-Quadruplex DNA

A solution of 5 μM KeTEL01 in 20 mM phosphate buffer, 100 mM KCl, pH 7.0was placed in a quartz cuvette and the UV/VIS spectrum determined. Analiquot of a solution of [d(TTAGGGT)]4 (SEQ ID No. 6) was added to thecuvette to afford a final concentration of 50 μM. The cuvette wasquickly inverted several times and placed in the spectrophotometer. Theabsorption of the sample at 488 nm was continuously monitored for 3hours, during which time, the absorption decreased in a multiexponentialfunction. The time required for the absorption at 488 nm to reachone-half of its equilibrium value was 60 min.

Example 8 Telomerase Inhibition by UT-SK-02 (DiethylthiocarbocyanineIodide)

Using the DOCK screening methods above, the carbocyanine group ofcompounds were identified as potential G-quadruplex interactive agents.A number of these compounds were assayed using the DNA Synthesis ArrestAssay described in example 5.4. Each compound was assayed at aconcentration of 20 μM. The results of this study are summarized inTable 1 ahead:

TABLE 1 Stop Stop Compound (F + P)_(rel)* (P/T)_(rel)** IBT-129A 68% 91%UT-SK-001 86% 83% UT-SK-002 88% 139%  UT-SK-003 29% 32% UT-SK-004 71%88% UT-SK-006 33% 12% *Relative amount of both full-length and pausedproducts. **Relative ratio of the amount of paused products as comparedto the total amount of products.

Of the compounds tested, only one, UT-SK-002 (diethylthiocarbocyanineiodide) demonstrated a specific interaction with G-quadruplex DNA, asindicated by a relative ratio of paused to total DNA product greaterthan 100% and a relative amount of DNA products, both paused and fulllength, that is close to 100%. In confirmatory tests, only this compoundinhibited telomerase, with an inhibition of 10-35% at a concentration of50 μM.

Example 9 Reduced Cellular Proliferation by Selected Compounds

The ability of these compounds to inhibit the proliferative capacity ofhuman cancer cells was determined by a standard MTT assay. Briefly,cells were incubated for 72 hours in the presence of variousconcentrations of compound, and the cell viability was determined bymonitoring the formation of a colored formazan salt of the tetrazoliumsalt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)by viable cells. KeTEL01 showed no cytotoxic effect up to the highestconcentrations tested (100 μM), whereas KeTEL03(N,N′-bis(2-dimethylaminoethyl)-3,4,90,10-perylenetetracarboxylica aciddiimide, see Example 1) showed good cytotoxicity in a variety of humancancer cell lines. Thus, KeTEL01, which is a selective G-quadruplexinteractive agent, has no acute (72 hr) cytotoxic effect, but thestructurally analogous KeTEL03, which does interact with double-strandedDNA as well as G-quadruplex, is cytotoxic under these assay conditions.

Cytotoxicity - IC₅₀ MTT Cell Lines KeTEL01 KeTEL03 MCF-7 >100 μM 18.4μM  BT-20 >100 μM 3.8 μM PC-3 >100 μM 3.8 μM Raji >100 μM 0.4 μM

Example 10 DNA Oligonucleotides

The DNA primer extension sequence P18 (5′-TAATACGACTCACTATAG3′) (SEQ IDNo. 12) and the template sequences shown in Table 1 were synthesizedusing a PerSeptive Biosystems Expedite 8909 synthesizer and purifiedwith denaturing polyacrylamide gels. The template DNA was diluted to 5ng/μL and dispensed into small aliquots.

Example 11 DMS Methylation Protection Assay

The ³²P-labeled PQ74 and HT4 templates were denatured by heating at 90°C. for 5 min and then cooled down to room temperature in 50 mM Tris-HClbuffer with or without 100 mM of K⁺. One microliter of 1:4ethanol-diluted DMS was added to 1 μg (300 μL) of annealed DNA. Aliquotswere taken at time points as indicated in the figures, and modificationreactions were stopped by adding ¼ volume of stop buffer containing 1 Mof β-mercaptoethanol and 1.5 M of sodium acetate. The modificationproducts were ethanol precipitated twice and treated with piperidine.After ethanol precipitation, the cleaved products were resolved on a 16%polyacrylamide gel.

Example 12 DNA Synthesis Arrest Assay

This assay is a modification of that described by Weitzmann andco-workers (Weitzmann et al., 1996). Briefly, primers (P18, 24 nM)labeled with [γ-³²P] were mixed with template DNA (12 nM)) in a Tris-HClbuffer (10 mM Tris, pH 8.0) containing K⁺ (5 mM for the PQ74 templateand 50 mM for the HT4 template) and denatured by heating at 90° C. for 5min. After cooling down to room temperature, BSU-1051 was added atvarious concentrations and incubated at room temperature for 15 min. Theprimer extension reactions were initiated by adding dNTP (finalconcentration 100 μM), MgCl₂ (final concentration 3 μM), and Taq DNApolymerase (2.5 U/reaction, Boehringer Mannheim). For sequencingreactions, the TaqTrack Sequencing System (Promega, Madison, Wis.) wasused. The sequencing reaction buffer was changed to 50 mM Tris-HCl, pH9.0, 10 mM MgCl, and 50 mM K⁺. The reactions were stopped by adding anequal volume of stop buffer (95% formamide, 10 mM EDTA, 10 mM NaOH, 0.1%xylene cyanol, 0.1% bromphenol blue). For the temperature-dependentstudies, the ligand concentration was fixed and the primer extensionreactions were carried out at the temperatures indicated in methods. Theproducts were separated on a 12% polyacrylamide sequencing gel. The gelswere then dried and visualized on a PhosphorImager (Molecular Dynamicsmodel 445 S1).

Example 13 Results The G-rich Regions of the PQ74 and HT-4 TemplatesForm Intermolecular G-quadruplex Structures in K⁺Buffer

To determine the nature of the G-quadruplex structures formed by thetemplate sequences used in this study (see Table 1), dimethylsulfate(DMS) was used to probe the accessibility of N7 of guanine in the DNAtemplates (Maxam and Gilbert, 1980). When the PQ74 template wasmethylated in 1×TE buffer, there was no apparent protection of anyguanine N7. However, with the exception of the first guanine in each ofthe four TTGGGG (SEQ ID No. 7) repeats, all the guanines in the G-richregion of the PQ74 template are protected from reacting with DMS in 100mM K⁺buffer, whereas guanines located outside the four repeats reactstrongly with DMS. This DMS protection pattern for the G-rich region ofthe PQ74 template in K⁺buffer suggests that only three guanines in eachof the four TTGGGG (SEQ ID No. 7) repeats are involved in G-tetradformation. This DMS reaction pattern is different from that observedpreviously by Henderson and co-workers (Henderson et al., 1990) with thed(TTGGGG)₄ (SEQ ID No. 7) G-quadruplex in which only the first guanineof the third repeat (corresponding to G9 in the PQ74 template) ishypersensitive to DMS methylation. On the basis of the results from theinventors' study, they propose a model for the G-quadruplex structureformed by the G-rich region of the PQ74 sequence consisting ofd(TTGGGG)₄ (SEQ ID No. 7). In this model, the first guanine of the firstrepeat is located in the 5′ overhang region and is therefore open to DMSmethylation. However, the first guanines of the second, third, andfourth repeats (G5, G9, and G13, respectively) are located in the loopregions of the G-quadruplex. Although the N7 groups of these three loopguanines are not involved in hydrogen bonds, steric inaccessibility mayprotect them from DMS methylation. The DMS footprinting pattern showsthat while they are partially protected from DMS methylation, thisprotection is less than that for the other guanines in the repeat.

The TTAGGG (SEQ ID No. 1) repeats in the G-rich region of the HT-4template also showed high DMS methylation protection in K⁺buffer. Inthis particular case, all three guanines in each repeat were almostevenly protected from methylation, indicating that all of them areinvolved in G-tetrad formation. This DMS methylation pattern isconsistent with the intramolecular G-quadruplex structure proposed byPatel and co-workers for the d[AG₃(T₂AG₃)₃] sequence based on NMRstudies (Wang and Patel, 1993).

(ii) BSU-1051 Binds to G-quadruplex DNA and Blocks DNA Synthesis in aConcentration Dependent Manner

Although it has been shown that G-quadruplex structures block primerextension by DNA polymerase in a K⁻dependent manner (Weitzmann et al.,1996), the inventors are unaware of any reports showing enhancedblockage by G-quadruplex —interactive agents. To determine if BSU-1051binding to G-quadruplex enhances the block to DNA synthesis, primerextension reactions were carried out in the absence and presence ofBSU-1051. Taq DNA polymerase primer extension on DNA templatescontaining four repeats of either TTGGGG (SEQ ID No. 7) (PQ74) or TTAGGG(SEQ ID No. 1) (HT4) in the presence of different concentrations ofBSU-1051 at 55° C. were performed. In these studies, K⁺was added at lowconcentrations (5 mM of K⁺for the PQ74 template and 20 mM of K for theHT4 template) in order to prevent overwhelming polymerase pausing due toformation of highly stable G-quadruplex structures. In the absence ofBSU-1051, there is only a slight pausing of the Taq DNA polymerase whenit reaches the 3′-end of the G-rich site on the template DNA at 55° C.However, upon increasing the concentration of BSU-1051, enhanced pausingis observed at the same site as that seen with low K⁺concentrations.This suggests that BSU-1051 enhances the polymerase pausing bystabilizing the G-quadruplex structure formed in the K⁺buffer. At highBSU-1051 concentrations, the inventors not only observed enhancedpausing at the 3′-end of the G-quadruplex site but also increasedpremature termination resulting from nonspecific interactions betweenBSU-1051 and the single-stranded template DNA. At a BSU-1051concentration of 100 μM, the primer extension is completely inhibiteddue presumably to nonspecific interactions between BSU-1051 and thesingle- and/or double-stranded DNA or between BSU-1051 and thepolymerase itself. In addition to the primary pausing site at thebeginning of the G-quadruplex site, two other secondary pausing sites atthe second and third G-rich repeats are observed at high BSU-1051concentrations. These pausings are probably induced by other structuresformed by this G-rich sequence. Given the fact that secondary pausingbeyond the first G-tetrad is not seen in the sequencing lanes thatcontain 50 mM K⁺, it is likely that these secondary pausings are causedby hairpin structures that are stabilized by BSU-1051 but not K⁺. Thissuggests that BSU-1051 has a relatively higher affinity for G-quadruplexDNA over other DNA secondary structures or single- and double-strandedDNA.

(iii) DNA Synthesis Arrest by the BSU-1051—G-quadruplex Complex Dependson the Stability of the G-quadruplex Structure

To further evaluate the ability of BSU-1051 to stabilize G-quadruplexDNA, Taq DNA polymerase primer extension reactions were carried out atfive different temperatures in the presence and absence of BSU-1051. Inthe absence of BSU-1051 polymerase pausing on the PQ74 templatecontaining four repeats of TTGGGG (SEQ ID No. 7) is almost lost ataround 65° C., which is presumably the melting point of the G-quadruplexstructure formed by this G-rich region in the template DNA. On the otherhand, in the presence of 20 μM BSU-1051, the G-quadruplex structure isfurther stabilized, and significant pausing is observed up to 74° C. Inthe HT4 template containing four repeats of TTAGGG (SEQ ID No. 1), inwhich the G-quadruplex structure formed is presumably less stable,pausing fades out at 55° C. in the absence of the ligand. However, inthe presence of BSU-1051, pausing is observed up to 65° C. Thus, forboth DNA sequences, ΔTm upon the addition of 20 μM BSU-1051 is about 20°C.

In order to confirm that the pausings seen result from the formation ofa G-quadruplex structure on the template DNA, certain guanines in thetemplates were substituted with 7-deaza-dG. Since N7 of guanine isinvolved in hydrogen bonding in the formation of a G-quadruplexstructure, substitution of guanine with 7-deaza-dG should preclude theformation of any G-quadruplex structure and allow for uninterruptedprimer extension on the template by Taq DNA polymerase in the presenceof either K⁺or BSU-1051. As shown in Table 1, two guanines in the TTAGGG(SEQ ID No. 1) repeat region of the HT4 template and four guanines inthe TTGGGG (SEQ ID No. 7) repeat region of the PQ74 template werereplaced with 7-deaza-dG. This change would allow the formation of nomore than two intramolecular G-tetrads and should lead todestabilization of the intramolecular G-quadruplex structure. The primerextension results with these 7-deaza-dG substituted templates indicatethat no significant pausing occurs in either template in the presence ofup to 20 mM of K⁺or at BSU-1051 concentrations of up to 50 μM. Thisresult provides strong support for the conclusion that BSU-1051 binds toand stabilizes intramolecular G-quadruplex DNA, leading to pronouncedDNA synthesis arrest at the G-quadruplex site in the original G-richtemplates.

Example 14 Discussion

G-rich sequences such as telomeric DNA and triplet DNA have beenreported to form parallel or antiparallel G-quadruplex structures in thepresence of monovalent cations such as Na⁺and K⁺. Williamson andco-workers observed very strong intramolecular UV cross-linking for thesequence d(TTGGGG)₄ (SEQ ID No. 7) in a 50 mM K⁺buffer (Williamson etal., 1989). Their results indicate that this sequence forms anintramolecular structure. Using DMS methylation, the inventors concludethat four repeats of TTGGGG (SEQ ID No. 7) or TTAGGG (SEQ ID No. 1)within a non-G-rich sequence are capable of forming an intramolecularG-quadruplex structure in K⁺buffer. Furthermore, the DMS methylationresults indicate that of the possible types of G-quadruplex structuresthat could be formed by d(TTGGGG)₄ (SEQ ID No. 7), a structureconsisting of three G-tetrads is the predominant species in 100 mM ofK⁺buffer. The proposed G-quadruplex structures formed by d(TTGGGG)₄ (SEQID No. 7) and d(TTAGGG)₄ (SEQ ID No. 7) repeats have diagonal loops, butalternative intramolecular G-quadruplex structures formed by foldoverhairpins consisting of three G-tetrads are also possible (Williamson,1994; Wang and Patel, 1995; Wang and Patel, 1994). However, theinventors could not differentiate between these two different types ofintramolecular G-quadruplex structures by the DMS methylation patternalone.

G-rich sequences that are capable of forming G-quadruplexes in vitro canbe found in telomeric sequences (Blackburn, 1991; Sundquist and Klug,1989; Kang et al., 1992), immunoglobulin switch regions (Sen andGilbert, 1988), the insulin gene (Hommond-Kosack et al., 1993), thecontrol region of the retinoblastoma susceptibility gene (Murchie andLilley, 1992), the promoter region of c-myc gene (Simonsson et al.,1998), fragile X syndrome triplet repeats (Nadel et al., 1995; Fry andLoeb, 1994), and HIV-1 RNA (Awang and Sen, 1993). It has been suggestedby Sen and Gilbert that telomeric DNA sequences may associate toinitiate the alignment of four sister chromatids by forming parallelguanine quadruplexes (Sen and Gilbert, 1990). Furthermore, the discoveryof G-quadruplex—forming sequences in the promoter region of certaingenes suggests that G-quadruplex structures may play a role in thetranscription regulation of these genes. Another possible role ofG-quadruplex DNA is the regulation of telomere length, since a telomericoverhang that forms a G-quadruplex structure would not be a goodsubstrate for telomerase (Henderson and Blackburn, 1989; Zahler et al.,1991). The inventors recently have demonstrated that BSU-1051 inhibitsprimer extension by telomerase only when the substrate (telomeric DNA)reaches four or more repeats in length (Sun et al., 1997). In thisreport, the inventors show that BSU-1051 is able to bind to andstabilize the intramolecular G-quadruplex structure formed by fourtelomeric repeats. Thus, it is reasonable to postulate that BSU-1051inhibits telomerase by interacting with its substrate(G-quadruplex—forming telomeric repeats) rather than telomerase itself.If G-quadruplex structures play important roles in other biologicalprocesses, then G-quadruplex—interactive compounds such as thosedescribed here, which stabilize these structures, may have a variety ofbiological effects. A series of 2,6-diamidoanthraquinones, includingBSU-1051, has been reported to moderate conventional cytotoxicity in arange of tumor cells (Collier and Neidle, 1988; Agbandje et al., 1992)and to inhibit human telomerase (Perry et al., 1998). The G-quadruplexbinding property of those compounds provides a possible mechanism fortheir action, although other mechanisms involving targeting of duplexDNA are also likely.

The inventors have recently proposed a model for a perylene—G-quadruplexcomplex based on NMR evidence (Fedoroff et al., 1998). By analogy withthis structure and that proposed for a TMPyP₄—G-quadruplex structure(Wheelhouse et al., 1998), it seems most likely that the binding site ofthe BSU-1051 is external to the lower G-tetrad and within the diagonalloop (see FIG. 5).

The block of DNA synthesis by G-quadruplex structures is not polymerasespecific. Woodford and co-workers showed that the K⁺ dependent DNAsynthesis arrest by G-quadruplex structures is similar for variouspolymerases (Woodword et al., 1994). The inventors have found that theBSU-1051—induced DNA synthesis arrest pattern is virtually identicalwhen Taq DNA polymerase, E. coli DNA polymerase I (Klenow fragment), orAMV reverse transcriptase is used. Given the fact that many G-rich DNAsequences are capable of forming G-quadruplexes in-vitro (particularlysome cancer related genes and sequences such as c-myc and telomeres),G-quadruplexs are targets for anticancer chemotherapy. The DNA synthesisstop assay described in this report provides a simple and rapid methodfor the identification of G-quadruplex—interactive agents as leadcompounds. This polymerase stop assay also allows an internal comparisonfor the relative binding of potential G-quadruplex—interactive compoundswith single and double-stranded DNA targets. This is an importantcomparison that may provide clues as to the relative cytotoxicity ofthese compounds.

The inventors have successfully used the present assay in theidentification and characterization of other G-quadruplex—interactivecompounds that are also telomerase inhibitors (Fedoroff et al., 1998;Wheelhouse et al., 1998). This assay can be used to identify otherG-quadruplex-interactive compounds with potential clinical utility.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Agbandje, Jenkins, McKerma, Reszka, Neidle, “Anthracene-9,10-diones aspotential anticancer agents. Synthesis, DNA binding, and biologicalstudies on a series of 2,6-disubstituted derivatives,” Med. Chem.,35:1418-1429, 1992.

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Chen, Kuntz, Shafer, “Spectroscopic recognition of guanine dimerichairpin quadruplexes by a carbocyanine dye,” Proc. Natl. Acad. Sci.U.S.A., 93:2635-2639, 1996.

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Haq, Ladbury, Chowdry, Jenkins, “Molecular anchoring of duplex andtriplex DNA by disubstituted anthracene-9/10-diones: calorimetric, UVmelting, and competition dialysis studies,” J. Am. Chem. Soc.,118:10693-10701, 1996.

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12 1 6 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 ttaggg 6 2 22 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 2 agggttaggg ttagggttag gg 22 3 7DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 3 taagggt 7 4 8 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 4 ttagggtt 8 5 7 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 5 aatgggt 7 6 7 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer6 ttagggt 7 7 6 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 7 ttgggg 6 8 22 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 8 agggttagggttagggttag gg 22 9 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 9 taatacgact cactatag 18 10 80 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 10tccaactatg tatacttggg gttggggttg gggttggggt tggggttagc ggcacgcaat 60tgctatagtg agtcgtatta 80 11 39 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 11 catggtggtt tgggttaggg ttagggttagggttaccac 39 12 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 12 taatacgact cactatag 18

What is claimed is:
 1. A method of identifying a telomerase inhibitorcomprising: a) contacting a compound with DNA G-quadruplex; and b)determining the melting point of the DNA (3-quadruplex wherein acompound exhibiting an increase in melting point of said quadruplex,relative to unbound DNA G-quadruplex, is indicated to inhibit telomeraseactivity.